Method for treating ifnalpha related conditions

ABSTRACT

An immunogenic product including IFNα coupled to a carrier protein molecule is capable to induce in vivo anti-IFNα antibodies and is useful in treating IFNα related conditions.

FIELD OF INVENTION

The present invention relates to an immunogenic vaccine and its use fortreating IFNα related conditions such as systemic lupus erythematosus.

BACKGROUND OF INVENTION

The IFN type I family includes IFNα, IFNβ, IFNδ, IFN1, IFNκ, IFNτ, andIFNω. The predominant forms are IFNα, of which 13 closely relatedproteins are described in humans, and the single IFNβ. Despite the factthat different IFN type I forms may promote different biologicalresponses, all IFN type I are structurally related (their genes lackintrons and are located on the short arm of chromosome 9) and signalthrough the same receptor subunits (Van Boxel-Dezaire et al., Immunity2006; 25:361-372).

The interest on the relationship between IFN type I and autoimmunedisorders is nowadays increasing, since the signs of its induction, theso-called interferon signature, have been recently reported in patientssuffering from different autoimmune diseases (Baccala et al. Immunol Rev2005; 204:9-26). In fact, due to its immune-modulator effects, IFN typeI seems to be involved in several pathogenic pathways of variousautoimmune conditions.

The paradigm of IFN type I pathogenic relevance in autoimmunity issystemic lupus erythematosus (SLE). SLE is a chronic disease,characterized by a multi-organ involvement, due to a paradoxical damageof organs caused by autoantibodies directed to self-antigens. Theetiology of SLE is complex, involving both genetic and environmentalfactors. The serum level of IFNα in SLE has been shown to correlate withthe severity of the disease (Dall'era et al. Ann Rheum Dis 2005;64:1692-7). Sjögren's syndrome (SS), also known as sicca syndrome, is achronic, systemic, autoimmune condition which affects the exocrineglands, particularly the salivary and lachrymal glands. Elevated IFNαactivity has also been observed in the serum of patients suffering fromthis disease. Finally, other conditions such as diabetes, rheumatoidarthritis, scleroderma, vasculitis and autoimmune thyroiditis have alsobeen shown to be associated with high levels of IFNα.

Sedaghat et al. also recently suggested that type 1 IFN may play a rolein CD4⁺ T cells depletion in HIV⁺ patients as they showed that type 1IFN affect the steady state of normal CD4⁺ T cells dynamics by shiftingthe balance towards Th1 effectors that are short lived cells instead oflong-lived memory T cells (Sedaghat et al. J. Virol. 2008, 82(4):1870-1883). This was confirmed in Mandl et al., where it is suggested todiminish the IFNα production by plasmacytoid dendritic cells toameliorate the pathological immune activation (Mandl et al. Nat. Med.2008).

Moreover, administration of IFNα has been reported to exacerbateunderlying disease in patients with psoriasis, autoimmune thyroiditisand multiple sclerosis and to induce an SLE like syndrome in patientswithout a previous history of autoimmune disease.

Therefore, there is a need for an agent that inhibits IFNα activity.

Passive immunization with monoclonal neutralizing antibodies iscurrently being tested in clinical trials with rontalizumab andsifalimumab for the treatment of SLE. However, said therapy presents thedrawbacks of targeting only one subset of the 13 for IFNα and the use ofpassively administrated monoclonal antibodies can be limited by theinduction of anti-drug antibodies. Said anti-drug antibodies mayneutralize or otherwise compromise the clinical effect of the drugs andcan also be associated with serious adverse events related tocross-reactivity with autologous proteins (De Groot et al. Trends.Immunol. 2007, 28(11)).

The present invention thus provides a method for inhibiting IFNαactivity in vivo by administering a therapeutically effective amount ofan immunogenic product that allows an active immunization which canbreak immunological B cell tolerance and generate high titers ofpolyclonal neutralizing antibodies against IFNα and its use for treatingIFNα related conditions.

SUMMARY

One object of the invention is an immunogenic product comprising IFNαcoupled to a carrier protein molecule for use in preventing or treatingan IFNα related condition in a subject in need thereof, wherein thetherapeutically effective amount of the immunogenic product to beadministrated to the subject is more than 30 mcg of immunogenic productper administration, preferably at least 60 mcg.

In one embodiment of the invention, the administration of thetherapeutically effective amount of the immunogenic product prevents theoccurrence of symptoms of a disease linked to an over-production ofIFNα.

In another embodiment of the invention, the administration of thetherapeutically effective amount of the immunogenic product prevents theflare of a disease linked to an over-production of IFNα.

In another embodiment of the invention, the IFNα related conditionscomprise systemic lupus erythematosus, rheumatoid arthritis,scleroderma, Sjögren syndrome, vasculitis, HIV, type I diabetes,autoimmune thyroiditis and myositis.

In another embodiment of the invention, the therapeutically effectiveamount of the immunogenic product to be administrated to the subject isfrom 35 mcg to 1000 mcg of immunogenic product per administration,preferably from 60 mcg to 1000 mcg.

In another embodiment of the invention, the immunogenic product isadministrated to the subject at least twice in a month.

In another embodiment of the invention, the immunogenic product isfurther administrated to the subject at least once every three months.

In another embodiment of the invention, the immunogenic product isfurther administrated to the subject when, in a serum sample obtainedfrom the subject, the amount of anti-IFNα antibodies is undetectable.

In another embodiment of the invention, the immunogenic product isstrongly inactivated, which means that the product shows less than 5% ofantiviral activity in the conditions of TEST B.

In another embodiment of the invention, the immunogenic product iscapable of neutralizing the antiviral activity of IFNα in the conditionsof TEST C.

In another embodiment of the invention, the immunogenic productcomprises at least one subtype of IFNα.

In another embodiment of the invention, the subtype of IFNα is IFNα 2band the carrier protein molecule is KLH.

In another embodiment of the invention, the immunogenic product is avaccine, preferably in the form of an emulsion.

Another object of the invention is a unit dosage form comprising morethan 30 mcg of an immunogenic product comprising IFNα coupled to acarrier protein molecule as defined here above.

Another object of the invention is a medical device comprising more than30 mcg of an immunogenic product comprising IFNα coupled to a carrierprotein molecule as defined here above.

Another object of the invention is a kit comprising at least one vialcontaining more than 30 mcg, preferably at least 60 mcg, of animmunogenic product comprising IFNα coupled to a carrier proteinmolecule as defined here above, at least one vial containing adjuvant,and means for contacting said immunogenic product to the adjuvant, andfor emulsifying the mixture of the aqueous solution with the adjuvant.

In one embodiment, the kit of the invention comprises

-   -   at least one vial containing more than 30 mcg, preferably at        last 60 mcg, of an immunogenic product comprising IFNα coupled        to a carrier protein molecule according to the invention, and        means for solubilizing said immunogenic product, preferably in        an aqueous solution, or    -   at least one vial containing a solution preferably an aqueous        solution, comprising more than 30 mcg, preferably at least 60        mcg, of an immunogenic product comprising IFNα coupled to a        carrier protein molecule according to the invention, and    -   at least one vial containing adjuvant, and means for contacting        said solution to the adjuvant, and for emulsifying the mixture        of the solution with the adjuvant.

Definitions

As used herein, the term “interferon α” or “IFNα” refers to IFN alphaproteins encoded by a functional gene of the interferon alpha gene locuswith 75% or greater sequence identity to IFN alpha 1 (Genbank numberNP_076918 or protein encoded by Genbank number NM_024013). Examples ofhuman IFN alpha subtypes include IFN alpha 1 (Genbank number NP_076918),alpha 2a (Genbank number ITF_A), alpha 2b (Genbank number AAP20099),alpha 4 (Genbank number NP_066546), alpha 5 (Genbank number P01569),alpha 6 (P05013), alpha 7 (Genbank number P01567), alpha 8 (Genbanknumber P32881), alpha 10 (Genbank number P01566), alpha 14 (Genbanknumber P01570), alpha 16 (Genbank number NP_002164), alpha 17 (Genbanknumber P01571) and alpha 21 (Genbank number NP_002166). Examples ofnonhuman mammal IFNa subtype may be found in Genbank as well known bythe person skilled in the art (for review see Pestka et al Immunologicalreviews 2004, 202:8-32).

As used herein, the term “immune response” refers to the action, forexample of lymphocytes, antigen presenting cells, phagocytic cells andmacromolecules produced by the above cells or the liver (includingantibodies, cytokines and complement).

As used herein, an antibody that “inhibits the biological activity” or“neutralizes the biological activity” of IFNα is intended to refer to anantibody that inhibits the activity of that cytokine by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, or 80% or more, as compared to the levelof activity of the cytokine in the absence of the antibody, for exampleby using a functional assay such as those described in the Examples.

As used herein, the term “carrier protein molecule” refers to a proteinor a peptide of at least 15 amino acids long which, when partiallycovalently being associated to the IFNα molecule for formingheterocomplexes, allows for a large number of antigens of IFNα to bepresented to the B lymphocytes.

As used herein, the term “subject” includes any human or nonhumanmammals such as primates, dogs, cats, horses, sheep . . .

As used herein, the term “patient” refers to a subject that is affectedby an IFNα related condition.

As used herein, the term “effective amount” refers to an amountsufficient to cause a beneficial or desired clinical result (e.g.improvement in clinical condition).

As used herein, the term “treatment” or “treating” refers to clinicalintervention in an attempt to alter the natural course of a disease ofthe subject or patient to be treated, and may be performed either forprophylaxis or during the course of clinical pathology. Desirableeffects include, but are not limited to, preventing occurrence orrecurrence of disease, alleviating symptoms, suppressing, diminishing orinhibiting any direct or indirect pathological consequences of thedisease, lowering the rate of disease progression, ameliorating orpalliating the disease state, and causing remission, maintainingremission state or improved prognosis.

DETAILED DESCRIPTION

Although regulators of IFNα occur naturally in the body, their capacityto regulate the cytokine levels in diseases such as SLE and SS appearsto be insufficient. The aim of the anti-IFNα therapeutic immunization ofthe invention is to raise antibody levels against the cytokine, whileenhancing their affinity and neutralizing activity, resulting in thereduction of the excess cytokine and inhibiting its pathogenic effects,without interfering with other metabolic and physiological processes.

One object of the present invention is a method for treating an IFNαrelated condition in a subject in need thereof, comprising administeringto the subject a therapeutically effective amount of an immunogenicproduct comprising IFNα coupled to a carrier protein molecule, whereinsaid therapeutically effective amount is more than 30 mcg (μg) ofimmunogenic product per administration.

In one embodiment of the invention, said therapeutically effectiveamount is at least 60 mcg (μg) of immunogenic product peradministration.

In one embodiment of the invention, the therapeutically effective amountof the immunogenic product per administration is from more than 30 mcg,preferably more than 60 mcg to 1000 mcg. In another embodiment of theinvention, the therapeutically effective amount of the immunogenicproduct per administration is from more than 30 mcg, preferably morethan 60 mcg to 750 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from more than 30 mcg, preferably more than 60 mcg to500 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is frommore than 30 mcg, preferably more than 60 mcg to 450 mcg. In anotherembodiment of the invention, the therapeutically effective amount of theimmunogenic product per administration is from more than 30 mcg,preferably more than 60 mcg to 400 mcg. In another embodiment of theinvention, the therapeutically effective amount of the immunogenicproduct per administration is from more than 30 mcg, preferably morethan 60 mcg to 350 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from more than 30 mcg, preferably more than 60 mcg to300 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is frommore than 30 mcg, preferably more than 60 mcg to 250 mcg. In anotherembodiment of the invention, the therapeutically effective amount of theimmunogenic product per administration is from more than 30 mcg,preferably more than 60 mcg to 200 mcg. In another embodiment of theinvention, the therapeutically effective amount of the immunogenicproduct per administration is from more than 30 mcg, preferably morethan 60 mcg to 150 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from more than 30 mcg, preferably more than 60 mcg to100 mcg.

In another embodiment of the invention, the therapeutically effectiveamount of the immunogenic product per administration is from 35 mcg to1000 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is from35 mcg to 750 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from 35 mcg to 500 mcg. In another embodiment of theinvention, the therapeutically effective amount of the immunogenicproduct per administration is from 35 mcg to 450 mcg. In anotherembodiment of the invention, the therapeutically effective amount of theimmunogenic product per administration is from 35 mcg to 400 mcg. Inanother embodiment of the invention, the therapeutically effectiveamount of the immunogenic product per administration is from 35 mcg to350 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is from35 mcg to 300 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from 35 mcg to 250 mcg. In another embodiment of theinvention, the therapeutically effective amount of the immunogenicproduct per administration is from 35 mcg to 200 mcg. In anotherembodiment of the invention, the therapeutically effective amount of theimmunogenic product per administration is from 35 mcg to 150 mcg. Inanother embodiment of the invention, the therapeutically effectiveamount of the immunogenic product per administration is from 35 mcg to100 mcg.

In another embodiment of the invention, the therapeutically effectiveamount of the immunogenic product per administration is from 60 mcg to1000 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is from60 mcg to 750 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from 60mcg to 500 mcg. In another embodiment of theinvention, the therapeutically effective amount of the immunogenicproduct per administration is from 60 mcg to 450 mcg. In anotherembodiment of the invention, the therapeutically effective amount of theimmunogenic product per administration is from 60 mcg to 400 mcg. Inanother embodiment of the invention, the therapeutically effectiveamount of the immunogenic product per administration is from 60 mcg to350 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is from60 mcg to 300 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from 60 mcg to 250 mcg. In another embodiment of theinvention, the therapeutically effective amount of the immunogenicproduct per administration is from 60 mcg to 240 mcg. In anotherembodiment of the invention, the therapeutically effective amount of theimmunogenic product per administration is from 60 mcg to 200 mcg. Inanother embodiment of the invention, the therapeutically effectiveamount of the immunogenic product per administration is from 60 mcg to150 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is from60 mcg to 120 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from 60 mcg to 100 mcg.

In another embodiment of the invention, the therapeutically effectiveamount of the immunogenic product per administration is from 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,360, 370, 380, 390 mcg to 400 mcg.

In another embodiment of the invention, the therapeutically effectiveamount of the immunogenic product per administration is from 60 mcg to240 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is 60mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is 120mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is 240mcg.

In one embodiment, the therapeutically effective amount corresponds toan amount of total proteins determined using a Bradford protein assay aswell known in the art.

In one embodiment of the invention, the subject to be treated isadministrated at least twice in a month with the therapeuticallyeffective amount of immunogenic product as described here above.

In another embodiment of the invention, the subject to be treated isadministrated two times in 1 month with the therapeutically effectiveamount of immunogenic product as described here above. In thisembodiment, the subject may be administrated once at day 0 and thesecond time between day 7 and day 28. In another embodiment, the subjectmay be administrated once at day 0 and the second time between day 7 andday 21. In one embodiment, the subject is administrated once at day 0and the second time at day 28.

In another embodiment of the invention, the subject to be treated isadministrated three times in 1 month with the therapeutically effectiveamount of immunogenic product as described here above. In thisembodiment, the subject to be treated may be administrated once at day0, the second time between day 7 and day 14 and the third time betweenday 21 and day 28. In one embodiment, the subject is administrated onceat day 0, the second time at day 7 and the third time at day 28.

In another embodiment of the invention, the subject to be treated isadministered four times in 3 months with the therapeutically effectiveamount of immunogenic product as described here above. In thisembodiment, the subject to be treated may be administered one at day 0,the second time between day 7 and day 14, the third time between day 21and day 28 and the fourth time between day 77 and day 84. In oneembodiment, the subject is administered once at day 0, the second timeat day 7, the third time at day 28 and the fourth time at day 84.

In another embodiment of the invention, the subject to be treated may befurther administrated once every three months with the therapeuticallyeffective amount of the immunogenic product as described here above.

In one embodiment of the invention, the subject to be treated isadministered three times in one month as described here above, and thenfurther administered once every three months with the therapeuticallyeffective amount of the immunogenic product as described here above.

In one embodiment of the invention, the subject to be treated isadministered four times in three month as described here above, and thenfurther administered once every three months with the therapeuticallyeffective amount of the immunogenic product as described here above.

In another embodiment of the invention, the subject to be treated may befurther administrated once every six months with the therapeuticallyeffective amount of the immunogenic product as described here above.

In one embodiment of the invention, the subject to be treated isadministered three times in one month or four times in three month asdescribed here above, and then further administered once every sixmonths with the therapeutically effective amount of the immunogenicproduct as described here above.

In another embodiment of the invention, the subject to be treated may befurther administrated once a year with the therapeutically effectiveamount of the immunogenic product as described here above.

In one embodiment of the invention, the subject to be treated isadministered three times in one month or four times in three month asdescribed here above, and then further administered once every year withthe therapeutically effective amount of the immunogenic product asdescribed here above.

In another embodiment of the invention, the subject to be treated may befurther administrated once every 5 years with the therapeuticallyeffective amount of the immunogenic product as described here above.

In one embodiment of the invention, the subject to be treated isadministered three times in one month or four times in three month asdescribed here above, and then further administered once every 5 yearswith the therapeutically effective amount of the immunogenic product asdescribed here above.

In another embodiment of the invention, the subject to be treated may befurther administrated once every 10 years with the therapeuticallyeffective amount of the immunogenic product as described here above.

In one embodiment of the invention, the subject to be treated isadministered three times in one month or four times in three month asdescribed here above, and then further administered once every 10 yearswith the therapeutically effective amount of the immunogenic product asdescribed here above.

In another embodiment of the invention, the subject to be treated may befurther administrated with the therapeutically effective amount of theimmunogenic product as described here above when the amount ofantibodies against IFNα is undetectable in a serum sample obtained fromthe subject.

In one embodiment of the invention, the subject to be treated isadministered three times in one month or four times in three month asdescribed here above, and then further administered with thetherapeutically effective amount of the immunogenic product as describedhere above when the amount of antibodies against IFNα is undetectable ina serum sample obtained from the subject.

Quantification of the amount of antibodies against IFNα in a serumsample may be carried out by conventional methods known in the art, suchas an ELISA anti-IFN.

One example of carrying out such method is the following:

-   -   coating a 96 wells plate with 100 ng of the subtype of IFNα used        for preparing the immunogenic product such as IFNα-2b and        incubate the plate overnight at 2° C.-8° C.,    -   blocking the plate with a blocking buffer during 90 min at 37°        C.,    -   incubating the plate with the serum sample and pool of naïve        sample during 90 min at 37° C.: the serum sample is typically        diluted in a two fold dilution series starting from dilution        200× to at least 8 dilutions,    -   incubating the plate with the labeled secondary antibody such as        a goat anti-human immunoglobulin conjugated to HRP,    -   developing the complex with an o-phenylenediamine        dihydrochloride (OPD) substrate solution. After stopping the        enzymatic reaction, the intensity of the resulting color is        determined by spectrophotometric methods at 492 nm.

The anti-IFN titer for each sample is expressed as the minimal dilutionfor which the mean OD value is higher than the cut-off value:

Cut-off value=Mean OD of the pool of naive serum×2.08

where the N cut-off value is equal to 2.08.

Then, the anti-IFN titer for each sample will be expressed as theminimal dilution for which the mean OD value is higher than the cut-offvalue. The first dilution being 200, patients are considered negative iftheir OD at 1/200 is inferior to the cut-off value (Mire-Sluis et al.2004 J. Immunol Meth. 289: 1-16).

In one embodiment of the invention, the subject to be treated issuffering from an IFNα related condition.

In another embodiment of the invention, the subject to be treatedpresents undetectable amount of anti-IFNα antibodies in the serum.

[Mechanism of Action]

The present invention also relates to an immunogenic product that isuseful for inducing an immune response in a mammal to whom saidimmunogenic product is administered, including a humoral immune responsewherein antibodies that neutralize the immmunosuppressive, apoptotic orangiogenic properties of the endogenous cytokine IFNα.

The present invention also relates to a method for inducing an immuneresponse in a mammal in need thereof, said method comprising theadministration of an immunogenic product as hereinabove described tosaid mammal. In one embodiment, said immune response includes a humoralimmune response wherein antibodies that neutralize theimmunosuppressive, apoptotic or angiogenic properties of the endogenouscytokine are induced.

In one embodiment of the invention, the immunogenic product is aninactivated but immunogenic cytokine derivative of IFNα chemicallycoupled to a T-helper stimulating foreign carrier protein such as forexample KLH. Said immunogenic product has the ability to disrupt B cellbut not T cell tolerance to IFNα. Helper T cell tolerance against selfis circumvented by linking IFNα to the foreign carrier protein.

B cells specific for IFNα are activated following antigen binding andendocytose the immunogenic product and carrier specific peptides arepresented via the Major Histocompatibility Complex (MHC) class IImolecules. This activation signal is not sufficient to induce B celldifferentiation in the case of a T dependent antigen but because B cellsprocess the self and the carrier antigens, T cell help can be given by Tcells specific for the self or the carrier protein. Since T cellselection is very stringent, there is no specific T cell activation forthe self antigen.

Dendritic cells (DC) can also take up the self antigen and the carriermolecule and present carrier specific peptides via their MHC class IImolecules. DCs are thus able to activate naïve T helper cells specificfor the carrier. The T helper cells are in turn able to providecarrier-specific T helper cells to B cells specific for the self antigenand to present carrier peptides on their MHC class II molecules.

T helper cells specific for the carrier interact with B cells specificfor the self antigen, eliciting a normal antibody response against theself antigen.

The immunogenic product is mainly used in vaccine compositions fortreating a disease linked to an over-production of IFNα.

More specifically, this invention relates to a method for treating adisease linked to an over-production of IFNα comprising a step ofadministering to the subject, a therapeutically effective amount of theimmunogenic product of the invention.

This invention also relates to a method for treating a disease linked toan over-production of IFNα comprising the administration of atherapeutically effective amount of the immunogenic product, wherein theadministration of the immunogenic product prevents the occurrence ofsymptoms of the disease.

The invention also relates to a method for treating a disease linked toan over-production of IFNα comprising the administration of atherapeutically effective amount of the immunogenic product, wherein theadministration of the immunogenic product prevents the flare of thedisease.

The invention also relates to a method for treating a disease linked toan over-production of IFNα comprising the administration of atherapeutically effective amount of the immunogenic product, wherein theadministration of the immunogenic product induces the production ofantibodies that neutralize the activity of endogeneous IFNα.

The invention also relates to a method for treating a disease linked toan over-production of IFNα comprising the administration of atherapeutically effective amount of the immunogenic product, wherein theadministration of the immunogenic product induces the neutralization ofthe activity of endogeneous IFNα.

Examples of disease linked to an over-production of IFNα include, butare not limited to systemic lupus erythematosus, rheumatoid arthritis,scleroderma, Sjögren syndrome, vasculitis, HIV, type I diabetes,autoimmune thyroiditis and myositis.

A further object of the invention consists of a method for inducing theproduction of antibodies that neutralize the activity of endogeneousIFNα in a subject, comprising a step of administering to said subject atherapeutically effective amount of the immunogenic product.

[The Immunogenic Product]

The immunogenic product as used in the invention comprises IFNα coupledto a carrier protein molecule such as KLH, wherein the immunogenicproduct is inactivated.

The immunogenic product as used in the invention is a complex between atleast one recombinant IFNα subtype and at least one carrier proteinmolecule such as for example KLH obtained by conjugation withglutaraldehyde and subsequent inactivation with formaldehyde.

In one embodiment of the invention, the carrier protein molecule may beany carrier molecule conventionally used in immunology such as KLH(Keyhole limpet hemocyanin), ovalbumin, bovine serum albumin (BSA),toxoid tetanos, toxoid diphteric B cholera toxin, mutant non toxicdiphtheria toxin (CRM197), neisseria meningitidis outer membrane proteinin outer membrane vesicles, non-typeable Haemophilus influenza outermembrane protein, pseudomonas aeruginosa toxin A, virus like particle(VLP) . . . In one preferred embodiment, said carrier is KLH.Preferably, the KLH starting product consists of a highly purified KLHextracted from the lymph of the marine gastropod mollusk Megathuracremulata. Naturally produced KLH generally consists of a di-decamerstructure which is a non covalent tubular assembly of 20 subunits.

In another embodiment of the invention, the recombinant IFNα subtype maybe any subtype among IFN alpha 1, alpha 2a, alpha 2b, alpha 4, alpha 5,alpha 6, alpha 7, alpha 8, alpha 10, alpha 14, alpha 16, alpha 17 andalpha 21.

Recombinant IFNα subtypes may be obtained by conventional methods knownin the art using the sequences from Genbank as described here above. Forexample, production of the recombinant IFNα subtype may be carried outby culturing cells containing an expression vector comprising the geneof the IFNα subtype and then harvesting the inclusion bodies and finallypurifying the IFNα subtype.

In one embodiment of the invention, the recombinant IFNα subtype is theIFNα 2b subtype.

In one embodiment of the invention, the immunogenic product comprises atleast the IFNα 2b subtype.

In one embodiment of the invention, the recombinant IFNα subtype is in aliquid solution, preferably a buffer solution having a pH ranging from3.5, preferably from 6 to 7.8.

In one embodiment, when the subject to be treated is a human, therecombinant IFNα used is human.

In one embodiment of the invention, the immunogenic product comprisesIFNα coupled to a carrier protein molecule such as for example KLH,wherein said immunogenic product is recognized by an anti-IFNα antibody.

The recognition of the immunogenic product by an anti-IFNα antibody maybe carried out by conventional methods known in the art such as asandwich ELISA anti-IFNα/carrier protein. The ELISA (TEST D) aredeveloped by any colorimetric means known in the art such as for exampleusing detection antibody labelled with biotin, a poly-streptavidin HRPamplification system and an o-phenylenediamine dihydrochloride substratesolution.

One example of said method is the following:

-   -   coating a plate with the capture antibody, such as for example a        rabbit polyclonal anti-KLH antibody,    -   blocking the plate with a blocking buffer (such as casein 2% in        PBS for example) during 90 min at 37° C.,    -   incubating during 90 min at 37° C. the plate with a dilution        series of the immunogenic product from 250 ng/ml to 8 two fold        dilutions or with negative controls such as KLH and IFNα,    -   incubating 90 min at 37° C. the plate with the detection        antibody such as for example a biotinylated anti-IFNα antibody,    -   incubating the plate with streptavidin-HRP during 30 min at        37° C. and developing the complex with an o-phenylenediamine        dihydrochloride (OPD) substrate solution furing 30 min. After        stopping the enzymatic reaction, the intensity of the resulting        color is determined by spectrophotometric methods at 490 nm.

When optical density of wells containing the immunogenic product is atleast 10 times superior to the optical density of wells containing thenegative control, the person skilled in the art considers that theimmunogenic product is recognized by an anti-IFNα antibody and that IFNαin the immunogenic product is coupled to the KLH.

In another embodiment of the invention, the immunogenic productcomprises IFNα coupled to a carrier protein molecule such as for exampleKLH, wherein said immunogenic product is strongly immunogenic, whichmeans that the product is capable of inducing antibodies anti-IFNα invivo in the conditions of hereunder tested TEST A.

Test A is carried out according to the following method:

0.3 to 10 μg of total proteins (as determined by a Bradford proteinassay) of the immunogenic product is injected in Balb/c mice of 6-8weeks twice in 30 days, preferably at day 0 and day 21. A serum sampleis obtained before immunization (pre-immune serum sample) and betweenday 30 and day 40 (test serum sample), preferably at day 31. An ELISAanti-IFNα is carried out as explained here above.

Briefly, a 96 wells plate is coated with 100 ng of the subtype of IFNαused for preparing the immunogenic product such as IFNα-2b and incubatedovernight at 2° C.-8° C. The plate is then blocked with a blockingbuffer during 90 min at 37° C. 100 μl of pre-immune sample at dilution1/2500 and a dilution series from 1/2500 up to 8 two fold dilutions ofthe serum samples (pre-immune and test) are added to the wells. Ananti-mouse immunoglobulins labeled secondary antibody such as an HRPconjugated antibody is finally added to the wells and the ELISA isdeveloped using any colorimetric means known in the art such as forexample an o-phenylenediamine dihydrochloride substrate solution.

When optical density of wells containing the test serum sample is atleast 2 times superior to the optical density of wells containing thepre-immune serum sample, the person skilled in the art considers thatthe immunogenic product is immunogenic, which means that it had inducedanti-IFNα antibodies in vivo.

In another embodiment of the invention, the immunogenic productcomprises IFNα coupled to a carrier protein molecule such as for exampleKLH, wherein the IFNα is strongly inactivated, which means that theproduct shows less than 5%, preferably less than 1% of antiviralactivity of IFNα in the conditions of hereunder cited TEST B. In oneembodiment, the immunogenic product of the invention at a concentrationof 500 ng/mL or more shows less than 5%, preferably less than 1% ofantiviral activity of IFNα at a concentration of 500 ng/mL or more inthe conditions of TEST B.

This assay is based on the protective effect of IFNα on the cytopathiceffect (CPE) of Vesicular Stomatitis Virus (VSV) on Madin-Darby BovineKidney (MDBK) cells. This assay may also be carried out using Hep-2C orA549 human cells and EMCV virus.

Test B is carried out according to the following method:

The immunogenic product and the recombinant IFNα subtype used forpreparing the immunogenic product (positive control) are diluted at atleast 500 ng/ml and at least 1000 U/ml respectively in Basal medium(RPMI supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 1 mMHepes). 50 μl of the immunogenic product and the positive control areplated in a 96 wells plate and diluted in a series of two fold dilutionsin the Basal medium. 2 10⁴ MDBK cells are added in each well in 50 μl ofCell medium (RPMI supplemented with 4% FBS, 2 mM glutamine, 1 mM sodiumpyruvate and 1 mM Hepes) and the plate is incubated overnight at 37° C.,5% CO₂. The virus is then diluted in Basal medium to at least 10 TCID₅₀(Tissue Culture Infection Dose 50: 10 times the dilution to kill 50% ofinfected cells). The plate is emptied and 100 μl of the diluted virus isadded. The plate is then incubated overnight at 37° C., 5% CO₂.

At the end of the culture, viability of the MDBK cells is assessed usingmethods well-known in the art. One example of said methods is thefollowing: 20 μl/well of a solution of MTS/PMS (100 μl MTS/5 μl PMS;Promega G5430) are added to the wells and the plate is incubated foranother 4 h at 37° C. 5% CO2. The plate is then read at 490 nm on aspectrophotometer.

The percentage of antiviral activity is calculated as following:

% antiviral activity=[OD_(product)−OD_(virus))/meanOD_(cells)−OD_(virus))]*100

OD_(product) stands for the optical density of well with the immunogenicproduct or with the positive control (IFNα subtype).

OD_(virus) stands for the optical density of control well with the virusonly.

OD_(cells) stands for the optical density of control well with IFNα andvirus.

The EC₅₀ value, corresponding to the amount of immunogenic productresulting in 50% inhibition of virus-mediated mortality, is determinedby interpolating the EC₅₀ value onto the x axis on aviability/concentration graph.

Comparing the EC₅₀ of the immunogenic product and the EC₅₀ of thepositive control (the recombinant IFNα subtype used for preparing theimmunogenic product) allows determining whether the immunogenic productshows less than 5%, preferably less than 1% of antiviral activity.

An Inactivation Factor EC_(50 product)/EC_(50 IFNα) can be calculated:when the immunogenic product shows less than 5%, preferably less than 1%of antiviral activity, the Inactivation Factor is more than 20,preferably more than 100.

In another embodiment of the invention, the immunogenic productcomprises IFNα coupled to a carrier protein molecule such as for exampleKLH, wherein the immunogenic product is capable of neutralizing theantiviral activity of IFNα in the conditions of hereunder cited TEST C.According to the invention, this assay is performed to evaluate theneutralizing capacity of the serum obtained from mice immunized with theimmunogenic product. The neutralizing capacity may be assessed byevaluating the cell viability in presence of the vesicular stomatitisvirus replicating in MDBK cells. This assay may also be carried outusing Hep-2C human cells and EMCV virus.

Test C is carried out according to the following method:

0.3 to 10 μg of total proteins (as determined by a Bradford proteinassay) of the immunogenic product is injected in Balb/c mice of 6-8weeks twice in 30 days, preferably at day 0 and day 21. A serum sampleis obtained before immunization (pre-immune serum sample) and betweenday 30 and day 40 (test serum sample), preferably at day 31.

25 μl of pre-immune and test serum samples are plated in a 96-well plateat a dilution of 1/200 up to 8 dilutions from 1/200. The positivecontrol (polyclonal anti-IFNα from PBL, Piscataway, N.J., ref. 31100-1)is typically diluted to be able to neutralize IFNα activity from 3125UI/well to 100 UI/well in Basal medium (RPMI supplemented with 2 mMglutamine, 1 mM sodium pyruvate and 1 mM hepes) and 25 μl were alsoplated in the plate.

25 U/well (final concentration) in 25 μl of basal medium of IFNα isadded to each well and the plate is incubated for 60 min at roomtemperature.

20000 MDBK cells in Assay medium (RPMI supplemented with 4% FBS, 2 mMglutamine, 1 mM sodium pyruvate, 1 mM hepes) are added to each well andthe plate is incubated overnight at 37° C., 5% CO₂.

The virus is diluted to at least 10 TCID₅₀ (10 times the dilution tokill 50% of infected cells) in virus medium (RPMI supplemented with 2 mMglutamine, 1 mM sodium pyruvate, 1 mM hepes). The plate is emptied and100 μl of virus is added to each well before incubation for 24 h at 37°C., 5% CO₂.

At the end of the culture, viability of the MBDK cells is assessed usingmethods well-known in the art. One example of said methods is thefollowing: 20 μl/well of a solution of MTS/PMS (100 μl MTS/5 μl PMS;Promega G5430) are added to the wells and the plate is incubated foranother 4 h at 37° C. 5% CO2. The plate is then read at 490 nm on aspectrophotometer.

The relative cell viability is calculated as following:

%=[OD_(sample)−OD_(virus))/OD_(IFN+virus))]*100

OD_(sample) stands for the optical density of well with the serumobtained from the mouse immunized with the immunogenic product or withthe positive control (polyclonal anti-IFN antibody).

OD_(virus) stands for the optical density of control well with the virusonly.

OD_(IFN+virus) stands for the optical density of control well with IFNαand virus.

The NC₅₀ value, corresponding to the dilution of serum resulting in 50%neutralization of virus-mediated mortality expressed as a dilutionfactor or neutralizing unit/ml, is determined by interpolating the NC₅₀value onto the x axis on a viability/concentration graph.

In TEST C, a result showing that the serum obtained from the mouseimmunized with the immunogenic product does not protect the MBDK cellsfrom mortality means that the immunogenic product has the capacity toinduce antibodies directed against IFNα that neutralize its antiviralactivity.

In one embodiment, the immunogenic product comprises IFNα coupled to acarrier protein molecule such as for example KLH, wherein theimmunogenic product is capable of neutralizing at least 50% of theantiviral activity of IFNα in the conditions of TEST C. In saidembodiment, the NC₅₀ can be calculated. If the dilution of serum is notcapable of neutralizing at least 50% of the antiviral activity of IFNαin the conditions of TEST C, the NC₅₀ of the product cannot becalculated.

In one embodiment of the invention, the immunogenic product comprisesIFNα coupled to a carrier protein molecule such as for example KLH,wherein the ratio IFNα/carrier in weight is ranging from 0.06 to 0.6.

In another embodiment of the invention, the immunogenic productcomprises IFNα coupled to a carrier protein molecule such as for exampleKLH, wherein the ratio IFNα/carrier is 0.1 to 0.5.

In another embodiment of the invention, the immunogenic productcomprises IFNα coupled to a carrier protein molecule such as for exampleKLH, wherein the ratio IFNα/carrier is 0.3.

In another embodiment of the invention, the immunogenic productcomprises IFNα coupled to a carrier protein molecule such as for exampleKLH, wherein the ratio IFNα/carrier is 0.05, 0.1, 0.2, 0.21, 0.22, 0.23,0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35,0.36, 0.37, 0.38, 0.39, 0.4, 0.5.

Said ratio may be calculated according to a method based on UV andfluorescence detection (Test E) as described in Example 10.

[Method for Obtaining the Immunogenic Product]

In one embodiment of the invention, the IFNα kinoid is obtainedaccording to the following method:

-   -   a) mixing together the at least one recombinant human IFNα        subtype and the at least one carrier protein molecule with        glutaraldehyde and blocking the reaction by adding a quenching        compound selected from (i) a reducing agent and (ii) an amino        acid selected from the group consisting of lysine and glycine        and mixture thereof,    -   b) removing compounds having a molecular weight of less than 10        kDa, or of less than 8 kDa    -   c) adding formaldehyde;    -   d) blocking the reaction with formaldehyde by adding a quenching        compound selected from (i) a reducing agent and (ii) an amino        acid selected from the group consisting of lysine and glycine        and mixture thereof,    -   e) collecting the said immunogenic product.

In one embodiment of step a), IFNα and the carrier protein molecule suchas for example KLH are firstly mixed together in the appropriateamounts, before adding glutaraldehyde.

In one embodiment, IFN α and KLH are mixed at step a) at aIFNα:subunitKLH molar ratio ranging from 10:1 to 40:1. In anotherembodiment, IFN α and KLH are mixed at step a) at a IFN α:subunitKLHmolar ratio ranging from 15:1 to 25:1. In another embodiment, IFN α andKLH are mixed at step a) at a IFN α:subunitKLH molar ratio ranging from20:1 to 25:1.

In one embodiment of step a), glutaraldehyde is used at a finalconcentration in the reaction mixture ranging from 1 mM to 250 mM,preferably from 20 mM to 30 mM, more preferably from 22.5 mM to 25 mM.In one embodiment of step a), glutaraldehyde is incubated with IFN α andKLH for a period of time ranging from 15 min to 120 min, preferablyabout 30, 35, 40, 45, 50, 60, 70, 80, 90 minutes. In one embodiment,glutaraldehyde is added at 22.5 mM during about 45 minutes.Advantageously, step a) of incubation with glutaraldehyde is performedat a temperature ranging from 18° C. to 37° C., preferably from 18° C.to 27° C.

According to an embodiment, the reaction with glutaraldehyde (step a) isstopped prior to removing compounds having a molecular weight of lessthan 10 kDa, (step b) by adding a quenching compound, preferably aquenching compound that is selected from (i) a reducing agent and (ii)an amino acid selected from the group consisting of lysine and glycineand mixture thereof.

The reducing agent may consist of any one of the reducing agents knownin the art which, due to their reducing properties, have the ability toreduce the remaining imine groupments generated during aldehydetreatment. The reducing agent may be selected from the group consistingof sodium borohydride, sodium cyanoborohydride.

According to an embodiment, in the embodiments wherein the saidquenching compound is an amino acid, the said amino acid consists ofglycine. In some embodiments of step b) where glycine and/or lysine areused for blocking the reaction with glutaraldehyde, the selected aminoacid is used at a final concentration in the reaction mixture rangingfrom 0.01 M to 1 M, preferably from 0.05 M to 0.5 M, and most preferablyfrom 0.08 M to 0.2 M, e.g. at 0.1 M as shown in the examples herein. Inan embodiment, incubation with the quenching compound is performed for aperiod of time ranging from 1 minute to 120 minutes, preferably from 5minutes to 60 minutes, e.g. for 30 minutes as shown in the examplesherein. In another embodiment, this step is performed at a temperatureranging from 18° C. to 30° C., preferably from 18° C. to 25° C.

At step b), the small compounds of less than 10 kDa that are present inthe reaction mixture are removed. These small compounds encompass mainlythe excess glutaraldehyde and the excess quenching compound moleculesthat have not reacted with IFN α nor KLH. Step b) may be performedaccording to any known technique which allows removing compounds of lessthan 10 kDa, which techniques include dialysis with a dialysis membranehaving a cut-off of 10 kDa or filtration using a filtration membranehaving a cut-off of 10 kDa. Illustratively, step b) may consist of astep of tangential flow filtration using a filtration membrane having acut-off of 10 kDa, as it is shown in the examples herein. The filtrationretentate, which is devoid of the undesirable small compounds, iscollected at the end of step b). If desired, step b) may comprise apreliminary step of removing the eventual compound aggregates present inthe reaction mixture obtained at the end of step b). The saidpreliminary step may consist of a conventional filtration step forremoving aggregates eventually present in suspension in a liquidsolution, e.g. a filtration step using an appropriate filtrationmembrane, e.g. a filtration membrane having a pore size of 0.2 μm.

In one embodiment of step c) of the method, formaldehyde is added at afinal concentration from 6 mM to 650 mM, preferably from 25 mM to 250mM. In one embodiment of step c) of the method, formaldehyde is addedfor a period of time from 1 h to 336 hours, preferably from 1 h to 144hours. In one embodiment, formaldehyde is applied at a finalconcentration of 50 to 100 mM, preferably 66 mM during 20 to 50 hours,preferably 40 hours.

At step c), incubation with formaldehyde is performed preferably at atemperature ranging from 30° C. to 40° C., e.g. at 37° C. as it is shownin the examples herein.

At step d) of the method, the reaction with formaldehyde is stopped byadding a quenching compound, preferably a quenching compound that isselected from (i) a reducing agent and (ii) an amino acid selected fromthe group consisting of lysine and glycine.

The reducing agent may consist in any one of the reducing agents knownin the art which, due to their reducing properties, reduce the remainingimine groupements generated during aldehyde treatment. The reducingagent may be selected from the group consisting of sodium borohydride,sodium cyanoborohydride. According to an embodiment, in the embodimentswherein the said quenching compound is an amino acid, the said aminoacid consists of glycine. In some embodiments of step b) where glycineand/or lysine are used for blocking the reaction with formaldehyde, theselected amino acid is used at a final concentration in the reactionmixture ranging from 0.01 M to 1.5 M, preferably from 0.05 M to 1 M, andmost preferably from 0.1 M to 0.2 M, e.g. at 0.1 M as shown in theexamples herein. In an embodiment, incubation with the quenchingcompound is performed for a period of time ranging from 5 minutes to 120minutes, preferably from 10 minutes to 60 minutes, e.g. for 30 minutesas shown in the examples herein. In another embodiment, this step isperformed at a temperature ranging from 18° C. to 30° C., preferablyfrom 18° C. to 25° C.

According to one embodiment of the method, just prior to collecting atstep e), removal of substances having a molecular weight of less than100 kDa may be performed by the skilled artisan by any technique knownin the art for removing substances having a molecular weight of morethan 100 kDa from a liquid solution. In a first embodiment, thetechnique used is a filtration step that is performed by using afiltration membrane having a cut-off value of at least 100 kDa, whichencompasses an ultrafiltration step or a tangential filtration step. Ina second embodiment, the technique used consists of a tangentialfiltration step using a filtration membrane having a cut-off value of atleast 100 kDa. In another embodiment, just prior to collecting at stepe), removal of substances having a molecular weight of less than 300 kDamay be performed by using a filtration membrane having a cut-off valueof at least 300 kDa.

[Composition, Emulsion and Vaccine Containing Such Emulsion]

This invention relates to a composition comprising the immunogenicproduct as described here above. This invention also relates to aformulation of the product of the invention, wherein the product iswithin an emulsion. Advantageously, the vaccine composition of theinvention comprises or consists of said emulsion. Such emulsioncomprises the immunogenic product of the invention, an oil and asurfactant or a mixture of at least one oil and at least one surfactant.Preferably, the oil or the mixture oil/surfactant is a pharmaceuticallyacceptable excipient. More preferably, the mixture of oil and surfactantis an adjuvant, even more preferably an immunoadjuvant. Preferredadjuvant is ISA 51. Another example of immunoadjuvant that may be usedis SWE (squalene-based oil-in-water emulsion). Another example ofimmunoadjuvant that may be used is SWE-a (squalane-based oil-in-wateremulsion). The emulsion of the invention may be a water-in-oil emulsionor an oil-in-water emulsion.

In another embodiment, the amount of the immunogenic product accordingto the invention is of more than 0.01% (w/w) and less than 1% (w/w) ofthe total weight of the said emulsion.

[Adjuvants]

The emulsion or the vaccine composition of the invention may compriseadjuvant, especially immunoadjuvants. In an embodiment, the amount ofadjuvant ranges from 0.00001% (w/w) to 1%, preferably 0.0001 to 0.1%,more preferably from 0.001 to 0.01% (w/w) of the total weight of thevaccine composition.

Any suitable adjuvant known by the skilled artisan may be used in thevaccine composition above, including oil-based adjuvants such as forexample Freund's Incomplete Adjuvant, mycolate-based adjuvants (e.g.,trehalose dimycolate), bacterial lipopolysaccharide (LPS),peptidoglycans (i.e., mureins, mucopeptides, or glycoproteins such asN-Opaca, muramyl dipeptide [MDP], or MDP analogs), MPL (monophosphoryllipid A), proteoglycans (e.g., extracted from Klebsiella pneumoniae),streptococcal preparations (e.g., OK432), Biostim™ (e.g., 01 K2), the“Iscoms” of EP 109 942, EP 180 564 and EP 231 039, aluminum hydroxide,saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils(such as arachid oil), liposomes, Pluronic™ polyols, the Ribi adjuvantsystem (see, for example GB-A-2 189 141), or interleukins, particularlythose that stimulate cell mediated immunity. An alternative adjuvantconsisting of extracts of Amycolata, a bacterial genus in the orderActinomycetales, has been described in U.S. Pat. No. 4,877,612.Alternatively, SWE (squalene 3.9%, span 0.47%, tween 80 0.47% in citratebuffer) and SWE-a (squalane 3.9%, span 0.47%, tween 80 0.47% in citratebuffer) may also be used. Additionally, proprietary adjuvant mixturesare commercially available. The adjuvant used will depend, in part, onthe recipient organism. The amount of adjuvant to administer will dependon the type and size of animal. Optimal dosages may be readilydetermined by routine methods.

Oil adjuvants suitable for use in water-in-oil emulsions may includemineral oils and/or metabolizable oils. Mineral oils may be selectedfrom BAYOU), MARCOL® and DRAKEOL®, including DRAKEOL® 6VR (SEPPIC,France). Metabolisable oils may be selected from SP oil (hereinafterdescribed), Emulsigen (MPV Laboratories, Ralston, NZ), Montanide264,266,26 (Seppic SA, Paris, France), as well as vegetable oils, suchas peanut oil and soybean oil, animal oils such as the fish oilssqualane and squalene, and tocopherol and its derivatives.

In addition, the adjuvant may include one or more wetting or dispersingagents in amounts of about 0.1 to 25%, more preferably about 1 to 10%,and even more preferably about 1 to 3% by volume of the adjuvant.Particularly preferred as wetting or dispersing agents are non-ionicsurfactants. Useful non-ionic surfactants includepolyoxyethylene/polyoxypropylene block copolymers, especially thosemarketed under the trademark PLURONIC® and available from BASFCorporation (Mt. Olive, N.J.). Other useful nonionic surfactants includepolyoxyethylene esters such as polyoxyethylene sorbitan monooleate,available under the trademark TWEEN 80®, or mannide monooleate. It maybe desirable to include more than one, e.g. at least two, wetting ordispersing agents in the adjuvant as part of the vaccine composition ofthe invention.

Suitable adjuvants may include but are not limited to surfactants knownby one skilled in the art, such as for example hexadecylamine,octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′-N-bis(2-hydroxyethyl-propane di-amine),methoxyhexadecyl-glycerol, and pluronic polyols; polanions, e.g., pyran,dextran sulfate, poly IC, polyacrylic acid, carbopol; peptides, e.g.,muramyl dipeptide, aimethylglycine, tuftsin, oil emulsions, alum, andmixtures thereof. Other potential adjuvants include the B peptidesubunits of E. coli heat labile toxin or of the cholera toxin. McGhee,J. R., et al., “On vaccine development,” Sem. Hematol., 30:3-15 (1993).

[Further Surfactants]

In the embodiments of a vaccine composition according to the inventioncomprising an emulsion, the vaccine composition preferably contains, inaddition to the combination of the immunogenic product and the one ormore oily immunoadjuvant substances, also one or more surfactant agents.Illustrative embodiments of surfactive agents include mannide monoleatesuch as MONTANIDE® 80 marketed by Arlacel (SEPPIC, France).

In an embodiment, the amount of surfactant agent ranges from 0.00001%(w/w) to 1%, preferably 0.0001 to 0.1%, more preferably from 0,001 to0.01% (w/w) of the total weight of the vaccine composition.

[Lyophilized Products]

According to an embodiment and for storage purposes, the product or thevaccine composition of the invention may be lyophilized. Vaccinecompositions may thus be presented in a freeze-dried (lyophilized) form.In said embodiment, the immunogenic product according to the inventionis combined with one or more lyophilisation auxiliary substances.Various lyophilisation auxiliary substances are well known by the oneskilled in the art. Lyophilization of auxiliary substances encompassessugars like lactose and mannitol.

In such embodiment where the vaccine composition consists of alyophilised composition for use as a liquid emulsion comprising asurfactant agent, the vaccine composition preferably comprises an amountof the immunogenic product according to the invention of more than 0.1%(w/w) and less than 10% (w:/w) of the total weight of the said vaccinecomposition.

[Stabilizers]

In some embodiments, the vaccine may be mixed with stabilizers, e.g. toprotect degradation-prone proteins from being degraded, to enhance theshelf-life of the vaccine, or to improve freeze-drying efficiency.Useful stabilisers are i.a, SPGA (Bovarnik et al; J. Bacteriology 59:509 (1950)), carbohydrates e.g. sorbitol, mannitol, trehalose, starch,sucrose, dextran or glucose, proteins such as albumin or casein ordegradation products thereof, mixtures of amino acids such as lysine orglycine, and buffers, such as alkali metal phosphates.

[Administration Route]

The vaccine compositions according to the invention may be administeredto the subject to be immunized by any conventional method including, byinjectable, e.g. intradermal, intramuscular, intraperitoneal, orsubcutaneous injection; or by topical, such as for example bytransdermal delivery. The treatment may consist of a single dose or aplurality of doses over a period of time.

[Dosage Form]

The forms suitable for injectable use may include sterile solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. The prevention againstcontamination by microorganisms can be brought about by adding in thevaccine composition preservatives such as various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal and the like. In many cases, it may be preferable toinclude isotonic agents, for example, sugars or sodium chloride, forreduce pain during injection. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminium monostearate andgelatine.

According to an embodiment, a lyophilized vaccine composition, of theinvention is solubilized in water for injection and gently mixed; thenan immunoadjuvant, preferably ISA 51, is added; the mixture is gentlymixed for emulsification and charged into a suitable syringe. Thisinvention thus also relates to a medical device, including a syringefilled or prefilled with a vaccine composition of the invention. Theemulsion is ideally prepared extemporaneously. However, the syringecontaining the emulsion can be stored less than 10 hours at 2-8° C. Inthis case, the emulsion should be allowed to warm up before injecting byfriction between the hands.

[Unit Dosage Range]

Another object of the invention is a dosage unit comprising an amount ofthe immunogenic product ranging from more than 30 mcg to 1000 mcg. Inanother embodiment, the dosage unit comprises an amount of theimmunogenic product ranging from 35 mcg to 1000 mcg. In anotherembodiment, the dosage unit comprises an amount of the immunogenicproduct ranging from 35 mcg to 750 mcg. In another embodiment, thedosage unit comprises an amount of the immunogenic product ranging from35 mcg to 500 mcg. In another embodiment, the dosage unit comprises anamount of the immunogenic product ranging from 35 mcg to 450 mcg. Inanother embodiment, the dosage unit comprises an amount of theimmunogenic product ranging from 35 mcg to 400 mcg. In anotherembodiment, the dosage unit comprises an amount of the immunogenicproduct ranging from 35 mcg to 350 mcg. In another embodiment, thedosage unit comprises an amount of the immunogenic product ranging from35 mcg to 300 mcg. In another embodiment, the dosage unit comprises anamount of the immunogenic product ranging from 35 mcg to 250 mcg. Inanother embodiment of the invention, the therapeutically effectiveamount of the immunogenic product per administration is from 60 mcg to1000 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is from60 mcg to 750 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from 60mcg to 500 mcg. In another embodiment of theinvention, the therapeutically effective amount of the immunogenicproduct per administration is from 60 mcg to 450 mcg. In anotherembodiment of the invention, the therapeutically effective amount of theimmunogenic product per administration is from 60 mcg to 400 mcg. Inanother embodiment of the invention, the therapeutically effectiveamount of the immunogenic product per administration is from 60 mcg to350 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is from60 mcg to 300 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from 60 mcg to 250 mcg. In another embodiment of theinvention, the therapeutically effective amount of the immunogenicproduct per administration is from 60 mcg to 240 mcg. In anotherembodiment of the invention, the therapeutically effective amount of theimmunogenic product per administration is from 60 mcg to 200 mcg. Inanother embodiment of the invention, the therapeutically effectiveamount of the immunogenic product per administration is from 60 mcg to150 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is from60 mcg to 120 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from 60 mcg to 100 mcg. In another embodiment, thedosage unit comprises an amount of the immunogenic product ranging from35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,330, 340, 350, 360, 370, 380, 390 to 400 mcg.

In another embodiment, the dosage unit comprises an amount of theimmunogenic product ranging from 60 mcg to 240 mcg. In anotherembodiment, the dosage unit comprises an amount of the immunogenicproduct ranging from 60 mcg to 120 mcg.

In another embodiment, the dosage unit comprises an amount of theimmunogenic product of 60 mcg. In another embodiment, the dosage unitcomprises an amount of the immunogenic product of 120 mcg. In anotherembodiment, the dosage unit comprises an amount of the immunogenicproduct of 240 mcg.

[Kit and Medical Device]

This invention also pertains to a kit comprising:

-   -   1 vial (Vial Number 1) containing the immunogenic product of the        invention, typically of 3 mL;    -   1 vial (Vial Number 2) containing adjuvant, preferably ISA51;        this vial is capable of containing 3 mL of adjuvant and may be a        container of 8 mL;    -   1 syringe, typically a Braun INJEKT-F® of 1 mL;    -   1 needle (Needle Number 1) for emulsion preparation; this needle        is preferably a 20 G needle;    -   1 needle (Needle Number 2) for injection, preferably        intramuscular injection; this needle is preferably a 23 G        needle.

This invention also pertains to a method for preparing a vaccine fromthe kit, comprising:

-   -   (1) pulling up 0.4 ml of adjuvant from Vial Number 2. Discharge        this syringe content into Vial Number 1 containing 0.4 ml of        immunogenic product.    -   (4) pumping up and down the total vial content a sufficient        number of times for emulsifying the content, typically 30 times        and finally pulling up the whole emulsion.

Prior to injection, Needle Number 1 is preferably switched for NeedleNumber 2 and air is purged from the syringe.

In one embodiment, said kit comprises:

-   -   1 vial (Vial Number 1) containing 0.4 ml of the immunogenic        product of the invention;    -   1 vial (Vial Number 2) containing at least 0.4 ml of adjuvant,        preferably ISA51;    -   1 syringe, typically a Braun INJEKT-F® of 1 mL;    -   1 needle (Needle Number 1) for emulsion preparation; this needle        is preferably a 20 G needle;    -   1 needle (Needle Number 2) for injection, this needle is        preferably a 23 G needle.

In another embodiment, the immunogenic product is in a lyophilized form.Therefore, the kit comprises:

-   -   1 vial (Vial Number 1) containing lyophilized product of the        invention, typically of 3 mL;    -   1 vial (Vial Number 2) containing water for injection typically        of 2 mL;    -   1 vial (Vial Number 3) containing adjuvant, preferably ISA51;        this vial is capable of containing 3 mL of adjuvant and may be a        container of 8 mL;    -   1 syringe, typically a Braun INJEKT-F® of 1 mL;    -   1 needle (Needle Number 1) for emulsion preparation; this needle        is preferably a 20 G needle;    -   1 needle (Needle Number 2) for injection, preferably        intramuscular injection; this needle is preferably a 23 G        needle.

This invention also pertains to a method for preparing a vaccine fromthe kit, comprising:

-   -   (1) injecting of water for injection from Vial Number 2 into the        Vial Number 1 by using the syringe connected to Needle number 1;    -   (2) rotating gently Vial Number 1 during 1-5 minutes until        complete solubilization of the preparation;    -   (3) with the same syringe and needle, pulling up adjuvant from        Vial Number 3. Discharge this syringe content into Vial Number        1.    -   (4) pumping up and down the total vial content a sufficient        number of times for emulsifying the content, typically 30 times        and finally pulling up the whole emulsion.

This invention also relates to the medical device which is the syringefilled or prefilled with the composition, emulsion or vaccine of theinvention.

In one embodiment, said syringe is a dual chamber syringe, wherein onechamber comprises a solution with the immunogenic product of theinvention and the other chamber comprises the adjuvant.

The invention also relates to a medical device comprising a vial or acarpule prefilled with the product of the invention or with the vaccinecomposition of the invention.

In one embodiment, the medical device comprises an amount of theimmunogenic product ranging from more than 30 mcg to 1000 mcg. Inanother embodiment, the medical device comprises an amount of theimmunogenic product ranging from 35 mcg to 1000 mcg. In anotherembodiment, the medical device comprises an amount of the immunogenicproduct ranging from 35 mcg to 750 mcg. In another embodiment, themedical device comprises an amount of the immunogenic product rangingfrom 35 mcg to 500 mcg. In another embodiment, the medical devicecomprises an amount of the immunogenic product ranging from 35 mcg to450 mcg. In another embodiment, the medical device comprises an amountof the immunogenic product ranging from 35 mcg to 400 mcg. In anotherembodiment, the medical device comprises an amount of the immunogenicproduct ranging from 35 mcg to 350 mcg. In another embodiment, themedical device comprises an amount of the immunogenic product rangingfrom 35 mcg to 360 mcg. In another embodiment, the medical devicecomprises an amount of the immunogenic product ranging from 35 mcg to250 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is from60 mcg to 1000 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from 60 mcg to 750 mcg. In another embodiment of theinvention, the therapeutically effective amount of the immunogenicproduct per administration is from 60mcg to 500 mcg. In anotherembodiment of the invention, the therapeutically effective amount of theimmunogenic product per administration is from 60 mcg to 450 mcg. Inanother embodiment of the invention, the therapeutically effectiveamount of the immunogenic product per administration is from 60 mcg to400 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is from60 mcg to 350 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from 60 mcg to 300 mcg. In another embodiment of theinvention, the therapeutically effective amount of the immunogenicproduct per administration is from 60 mcg to 250 mcg. In anotherembodiment of the invention, the therapeutically effective amount of theimmunogenic product per administration is from 60 mcg to 240 mcg. Inanother embodiment of the invention, the therapeutically effectiveamount of the immunogenic product per administration is from 60 mcg to200 mcg. In another embodiment of the invention, the therapeuticallyeffective amount of the immunogenic product per administration is from60 mcg to 150 mcg. In another embodiment of the invention, thetherapeutically effective amount of the immunogenic product peradministration is from 60 mcg to 120 mcg. In another embodiment of theinvention, the therapeutically effective amount of the immunogenicproduct per administration is from 60 mcg to 100 mcg. In anotherembodiment, the medical device comprises an amount of the immunogenicproduct ranging from 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 to 400 mcg.

In another embodiment, the medical device comprises an amount of theimmunogenic product ranging from 60 mcg to 240 mcg. In anotherembodiment, the medical device comprises an amount of the immunogenicproduct ranging from 60 mcg to 120 mcg.

In another embodiment, the medical device comprises an amount of theimmunogenic product of 60 mcg. In another embodiment, the medical devicecomprises an amount of the immunogenic product of 120 mcg. In anotherembodiment, the medical device comprises an amount of the immunogenicproduct of 240 mcg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Percentage of immunized patient serum samples showing IFNαneutralizing activity during interim report.

FIG. 2: Differential evolution of IFN-induced genes in treated—versusplacebo patients. Out of 11 patients displaying increased levels ofIFN-induced gene expression at baseline, 8 were treated with theimmunogenic product and 3 received placebo injections. The levels of 250IFN-induced genes showing the highest levels of over-expression in SLEpatients were evaluated using high-density microarrays. The results aredepicted as the mean log 2(level of expression at V1)−log 2(level ofexpression at V6). Thep value was calculated using a Student's t-test.

FIG. 3: Titers of IFN-binding antibodies in treated patients withpositive or negative IFN-signature at baseline versus placebo receivingpatients. Stars indicate p values<0.05.

FIG. 4: Differential evolution of IFN-induced genes in treated patientswith positive or negative IFN-signature at baseline versus placebopatients, between V10 and V0 or between V11 and V0. The results aredepicted as the mean Delta Log (Gene Expression). Stars indicate pvalues<0.05.

FIG. 5: Evolution of serum C3 values in treated patients with positiveIFN-signature at baseline and in placebo receiving patients. Starsindicate p values<0.04.

EXAMPLES Example 1 Preparation of the Immunogenic Product

Keyhole Limpet Hemocyanin (KLH) was extracted from the lymph of themarine gastropod mollusk Megathura crenulata and then purified under GMPcondition. Results from stability assays performed in storage conditionsat a temperature of 2-8° C. showed that the shelf life of the purifiedKLH is of 36 months at 2-8° C.

Recombinant human IFNα 2b was produced in E. coli under GMP conditions.

Batches of the product of the invention at 350 mg IFNα scale wereproduced using the manufacturing process developed below.

a) Conjugation with Glutaraldehyde

The filtered KLH is added to the IFNα 2b solution (IFNα 2b in 70 mMdi-sodium hydrogen phosphate pH 7.8) with a IFNα:KLH ratio of 20:1,(corresponding to a molar ratio of 20 monomer of IFNα for 1 subunit ofKLH) based on UV concentration. The conjugation is carried out withglutaraldehyde (added to reach 22.5 mM final concentration in thereaction medium) and borate pH 9 (added to reach 28.5 mM finalconcentration in the reaction medium), to obtain a pH of 8.5.

This solution at pH 8.5 is then mixed during 45 min at 23±2° C.

b) Quenching with Glycine

The reaction is quenched with Glycine 0.1 M during 30 min

c) First Tangential Flow Filtration (TFF 1)

The first TFF is performed with a Pall Minim II TFF system and apolyethersulfone membrane of 0.02 m² with a molecular weight cut off of10 kDa sanitized with 0.5 M NaOH and equilibrated with the workingbuffer (70 mM di-sodium hydrogen phosphate pH 7.8).

The quenched solution is then clarified by 0.22 μm-filtration. Theintermediate is diluted twice in the working buffer and then diafilteredby tangential flow filtration (TFF) and 12 volumes of working buffer.The retentate is harvested and is stored for less than 20 hours.

d) Inactivation with Formaldehyde

Formaldehyde is added to the retentate to reach a final concentration of66.6 mM using a peristaltic pump. The inactivation reaction is performedduring 40 hours in an incubator set to 37±2° C. with a daily mixing ofthe solution with a magnetic stirrer.

e) Quenching with Glycine

The reaction is then quenched with 0.1 M of Glycine during 30 min

f) Second Tangential Filtration (TFF 2)

The second TFF is performed with a Pall Minim II TFF system and apolyethersulfone membrane of 0.02 m² with a molecular weight cut off of100 kDa sanitized with 0.5 M NaOH and equilibrated with the formulationbuffer (70 mM di-sodium hydrogen phosphate pH 7.8).

The quenched solution is clarified by 0.2 m filtration. The intermediateis concentrated to have a starting tangential volume of 900 mL and nextfiltrated by TFF with 12 volumes of formulation buffer (70 mM phosphatebuffer) to eliminate the low molecular weight homopolymers of IFNα andthe non reactive reagents. The retentate is harvested and then dilutedto a theoretical concentration of 300 μg/mL based on concentrationdetermination by Bradford protein assay and then 0.2m-filtered to obtainthe immunogenic product of the invention.

Example 2 Antigenicity of the Product

A sandwich ELISA anti IFNα/KLH was carried out as following. Briefly, a96 wells plate was coated with the capture antibody: rabbit polyclonalanti-KLH antibody, and blocked with a blocking buffer (such as casein 2%in PBS for example) during 90 min at 37° C. The plate was incubatedduring 90 min at 37° C. the plate with a dilution series of theimmunogenic product from 250 ng/ml to 8 two fold dilutions or withnegative controls such as KLH and IFNα. A detection antibody such as forexample a biotinylated anti-IFNα antibody was then added for 90 min.Finally the plate was incubated with streptavidin-HRP during 30 min at37° C. and the complex developed with an o-phenylenediaminedihydrochloride (OPD) substrate solution during 30 min After stoppingthe enzymatic reaction, the intensity of the resulting color isdetermined by spectrophotometric methods at 490 nm.

This test confirmed that the product comprises IFNα that is antigenic,i.e. recognized by anti-IFNα antibody and that said IFNα is coupled toKLH.

Example 3 Immunogenicity of the Product (TEST A)

4 μg of total proteins of the product as determined by Bradford proteinassay were injected to 7 Balb/c mice of 6-8 weeks at day 0 and day 21.

At day 31, mice were bleeded and the sera were harvested.

An anti-IFNα ELISA was carried out on preimmune and harvested sera asfollowing:

-   -   a 96 wells plate was coated with 100 ng of IFNα-2b and incubated        overnight at 2° C.-8° C.,    -   a blocking buffer was added during 90 min at 37° C.,    -   the immunogenic product was added at a dilution of 1/2500 up to        at least 8 two fold dilutions and the plate was incubated during        90 min at 37° C.,    -   the plate was incubated with an anti-mouse immunoglobulin        labeled antibody such as an HRP conjugated antibody during 90        min at 37° C.,    -   the ELISA was developed with an o-phenylenediamine        dihydrochloride (OPD) substrate solution. After stopping the        enzymatic reaction, the intensity of the resulting color was        determined by spectrophotometric methods at 490 nm.

This test demonstrated that in the 7 mice, immunization with theimmunogenic product led to the presence of anti-IFNα antibodies titers.

Example 4 Residual Activity of the Product (TEST B)

This assay was based on the protective effect of IFNα on the cytopathiceffect (CPE) of Vesicular Stomatitis Virus (VSV) on Madin-Darby BovineKidney (MDBK) cells. The immunogenic product and the recombinant IFNα 2bused for preparing the immunogenic product (positive control) werediluted at at least 500 ng/ml and at least 1000 U/ml respectively inBasal medium (RPMI supplemented with 2 mM glutamine, 1 mM sodiumpyruvate, 1 mM Hepes). 50 μl of the immunogenic product and the positivecontrol were plated in a 96 wells plate and diluted in a series of twofold dilutions in the Basal medium. 2 10⁴ MDBK cells were added in eachwell in 50 μl of Cell medium (RPMI supplemented with 4% FBS, 2 mMglutamine, 1 mM sodium pyruvate and 1 min Hepes) and the plate wasincubated overnight at 37° C., 5% CO₂. The virus was then diluted inBasal medium to at least 10 TCID₅₀ (Tissue Culture Infection Dose 50: 10times the dilution to kill 50% of infected cells). The plate was emptiedand 100 μl of the diluted virus was added. The plate was then incubatedovernight at 37° C., 5% CO₂.

At the end of the culture, 20 μl/well of a solution of MTS/PMS (100 μlMTS/5 μl PMS; Promega G5430) were added to the wells and the plate wasincubated for another 4 h at 37° C. 5% CO2. The plate was then read at490 nm on a spectrophotometer.

The percentage of antiviral activity of the immunogenic product wascalculated and for the two batches of product tested, the antiviralactivity was less than 1% of the antiviral activity of IFNα.

Example 5 Neutralization Capacity of the Product (TEST C)

The neutralizing capacity of the product was assessed by evaluating thecell viability in presence of the vesicular stomatitis virus replicatingin MDBK cells.

4 μg of total proteins (as determined by a Bradford protein assay) ofthe immunogenic product were injected in Balb/c mice of 6-8 weeks at day0 and day 21. A serum sample was obtained before immunization(pre-immune serum sample) and at day 31 (test serum sample).

25 μl of pre-immune and test serum samples were plated in a 96-wellplate at a dilution of 1/200 up to 8 dilutions from 1/200. The positivecontrol (polyclonal anti-IFNα from PBL, Piscataway, N.J., ref. 31100-1)was diluted from 3125 UI/well to 100 UI/well in Basal medium (RPMIsupplemented with 2 mM glutamine, 1 mM sodium pyruvate and 1 mM hepes)and 25 μl were also plated in the plate.

25 U/well (final concentration) in 25 μl of basal medium of IFNα wasadded to each well and the plate is incubated for 60 min at roomtemperature.

20000 MDBK cells in Assay medium (RPMI supplemented with 4% FBS, 2 mMglutamine, 1 mM sodium pyruvate, 1 mM hepes) were added to each well andthe plate was incubated overnight at 37° C., 5% CO₂.

The virus was diluted to at least 10 TCID₅₀ (10 times the dilution tokill 50% of infected cells) in virus medium (RPMI supplemented with 2 mMglutamine, 1 mM sodium pyruvate, 1 mM hepes). The plate was emptied and100 μl of virus was added to each well before incubation for 24 h at 37°C., 5% CO₂.

At the end of the culture, 20 μl/well of a solution of MTS/PMS (100 μlMTS/5 μl PMS; Promega G5430) were added to the wells and the plate wasincubated for another 4 h at 37° C. 5% CO2. The plate was then read at490 nm on a spectrophotometer.

The NC was calculated for all the 7 test samples: mean NC=253789 IU/ml(SEM=172526), demonstrating that all serum comprised antibodiesanti-IFNα capable of neutralizing the antiviral activity of IFNα.

EXAMPLE 6 Examples of Compositions and Vaccine Comprising theImmunogenic Product

One illustrative composition comprising the immunogenic product isdescribed in Table 1.

TABLE 1 Components Quantity Product of the invention 160 μg di-sodiumphosphate 8.95 mg Disodium dihydrogen phosphate 805 μg Total volume 0.4ml

One illustrative vaccine comprising the immunogenic product is describedin Table 2.

TABLE 2 Emulsion Components Quantity Product of the invention 160 μgdi-sodium phosphate 8.95 mg Disodium dihydrogen phosphate 805 μg Drakeol6VR (mineral oil) 0.30 g Montanide 80 (mannide monooleate) 0.04 g Totalvolume 0.8 ml

Example 7 Clinical Trial

A clinical trial was carried out using the vaccine composition asdescribed in Table 2.

Study Design:

3 or 4 administrations of the product were performed at day 0, day 7 andday 28 or at day 0, day 7, day 28 and day 84 in adults subjected to SLE.

The following doses of the product were tested: 30 mcg, 60 mcg, 120 mcgand 240 mcg.

Study Population:

28 male or female patients aged between 18 and 50 years, with mild tomoderate SLE (SLEDAI 4-10), active disease despite receiving treatment.A normal control interferon gene signature was established in 48 healthyvolunteers. PBMC of 18 out of the 48 healthy volunteers were stimulatedin vitro with type I interferons in order to identify an interferonsignature on the high-density arrays. A SLE signature was established bycomparing the signatures between healthy volunteers and SLE patients atbaseline.

An interim analysis was performed in the patients enrolled in the firstthree groups, i.e. having received the 30, 60 or 120 mcg doses orplacebo.

TABLE 3 Demographics for enrolled patients (interim analysis)

Summary 30 mcg 60 mcg 120 mcg Placebo Total Statistics (N = 3) (N = 6)(N = 6) (N = 5) (N = 20) Age (years) Mean (SD) 36.0 (9.85) 39.3 (3.98)34.2 (12.12) 38.6 (11.52) 37.1 (9.28) Median 33.0 38.0  32.5  43.0  37.5Sex, n(%) Male 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Female 3 (100.0)6 (100.0) 6 (100.0) 5 (100.0) 20 (100.0) Race, n(%) White-Caucasian 3(100.0) 6 (100.0) 6 (100.0) 5 (100.0) 20 (100.0) SLEDAI-2000 Mean 8.67(1.15) 7.50 (2.81) 6.00 (2.19) 8.80 (1.09) Median  8.00  8.50  6.00 8.00 Anti-ds DNA ab Mean (SD) 53.93 (58.22) 61.25 (113.46) 140.55(242.63) 88.70 (113.62) Median  33.10 15.45 23.60 40.90 DURATION OFDISEASE (YEARS) Mean (SD) 10.0 (2.18) 8.9 (8.82) 7.3 (5.99) 5.9 (4.75)7.9 (6.11) Median 11.0 6.1 6.1 3.6  6.4 CONCOMMITANT 100% 66.7% 83.3%100% CORTICOSTEROIDS

indicates data missing or illegible when filed

TABLE 4 Demographics for enrolled patients (final analysis) 30 μg 60 μg120 μg 240 μg Placebo Measure N = 3 N = 6 N = 6 N = 6 N = 7 Age (y) Mean± SD 36.0 ± 9.8  39.3 ± 4.0  34.2 ± 12.1 34.8 ± 10.8 40.1 ± 10.2 Median33 38 33 36 43 Range 28-47 35-46 19-50 21-46 20-50 Sex Female n (%) 3(100) 6 (100) 6 (100) 6 (100) 7 (100) Ethnicity White-Caucasian n (%) 3(100) 6 (100) 6 (100) 6 (100) 6 (85.7) Asian n (%) 0 (0.0) 0 (0.0) 0(0.0) 0 (0.0) 1 (14.3) Weight (kg) Mean ± SD 69.7 ± 11.9 67.8 ± 10.059.2 ± 7.4  70.0 ± 15.9 57.4 ± 14.8 Median 75 63 59 65 55 Range 56-7858-81 51-71 54-97 46-90 Height (cm) Mean ± SD 162.3 ± 6.4  165.0 ± 5.1 164.0 ± 5.5  162.8 ± 8.6  162.7 ± 5.8  Median 165  166  165  163  163 Range 155-167 159-170 156-172 152-172 153-170 Body mass index Mean ± SD26.6 ± 6.0  25.0 ± 4.3  22.1 ± 3.5  26.7 ± 7.2  21.7 ± 5.2  (kg/m²)Median 27 24 21 25 20 Range 21-32 21-32 18-28 20-40 17-33 Diseaseduration (y) Mean ± SD 9.9 ± 2.2 8.9 ± 8.8 7.2 ± 6.0 11.8 ± 8.4  6.5 ±4.0 Range  7-11  1-23  0-18  2-21  1-11 SLEDAI 2000 Mean ± SD 8.7 ± 1.27.5 ± 2.8 6.0 ± 2.2 6.0 ± 1.8 8.4 ± 1.1 index Range  8-10  4-10  4-104-8  7-10 Medications at baseline, n (%) Glucocorticoids n (%) 3 (100.0)4 (66.7) 5 (83.3) 3 (50.0) 6 (85.7) Aminoquinolines n (%) 0 (0.0) 4(66.7) 3 (50.0) 5 (83.3) 5 (71.4) Methotrexate n (%) 0 (0.0) 1 (16.7) 1(16.7) 1 (16.7) 1 (14.3) Azathiopine n (%) 0 (0.0) 1 (16.7) 1 (16.7) 1(16.7) 0 (0.0)

Results

Safety and Tolerability of the Vaccine

Two lupus flares have been reported as related SAEs. The first was inthe placebo group. The other occurred after the first injection of IFN-K240 mcg in a patient who had spontaneously stopped her corticosteroidtherapy two days after injection. This abrupt stopping ofcorticosteroids treatment likely participated to the occurrence of theflare. Regular interim safety analyses were performed by an independentsafety board. No clinically significant change in laboratory parametershas been detected (hematology, biochemistry, urine).

Immunogenicity of the Vaccine

Anti-IFNα antibody titers were measured by ELISA from serum samplesobtained from the patients.

An anti-IFNα ELISA was carried out as described here above.

Results show that anti-IFNα antibody titers were detected in all groupstreated with the immunogenic product starting on day 28.

Neutralization Activity of the Vaccine

The neutralization activity was assessed in vitro using the followingmethod: 50 μl of serum samples obtained from the patients sera wereplated in a 96-well plate at a dilution of 1/200 up to 8 dilutions from1/200.

The positive control (polyclonal anti-IFN from PBL Piscataway, N.J.,31100-1) was diluted from 100 ng/well to 3.125 ng/well and 50 μl wereadded to the plate. Dilutions were carried out in Basal medium (RPMIsupplemented with 2 mM glutamine, 1 mM sodium pyruvate and 1 mM hepes).

10 U/well (final concentration) of IFNα 2b were added to each well andthe plate was incubated for 60 min at room temperature.

30000 MDBK cells in Assay medium (RPMI supplemented with 4% FBS, 2 mMglutamine, 1 mM sodium pyruvate, 1 mM hepes) were added to each well andthe plate was incubated overnight at 37° C., 5% CO₂.

The virus was diluted to at least 10 TCID₅₀ (10 times the dilution tokill 50% of infected cells) in virus medium (RPMI supplemented with 2 mMglutamine, 1 mM sodium pyruvate, 1 mM hepes). The plate was emptied and100 μl of virus was added to each well before incubation for 24 h at 37°C., 5% CO₂.

At the end of the culture, 20 μl/well of a solution of MTS/PMS (100 μlMTS/5 μl PMS; Promega G5430) were added to the wells and the plate wasincubated for another 4 h at 37° C. 5% CO2. The plate was then read at490 nm on a spectrophotometer.

The results of interim report showed that none of the sera from patientstreated with 30 mcg of the immunogenic product presents anti-IFNαantibodies having a neutralizing capacity at day 168 after immunization,whereas the sera from patients treated with 60 mcg of the immunogenicproduct present anti-IFNα antibodies having a neutralizing capacity atday 168 (FIG. 1).

Moreover, the results of the final report showed that a neutralizingactivity was detected in 50% of subject treated with 60 μg or 120 μg ofthe immunogenic product, and in 80% of subjects treated with 240 μg ofthe immunogenic product (Table 5)

TABLE 5 Dose (mcg) Number of responder (%) NC50 median at peak (Dil-1)30 0 0 60 50 390 120 50 733 240 80 316

These results demonstrated that treatment with more than 30 mcg of theimmunogenic product is necessary for having an in vivo neutralization ofIFNα.

Example 8 Transcriptomic Analysis

PBMC were harvested at several time-points before and after injection ofthe immunogenic product. For this interim analysis, total RNA wasextracted at V1 (day 0) and V6 (day 38 after the first injection)samples, labeled according to standard Affymetrix protocol, andhybridized on GENECHIP® HGU133 Plus 2.0 arrays. Statistical analyseswere performed on GENESPRING® after RMA (Robust Microarray Analysis)normalization of the samples.

Unsupervized clustering algorithms were performed on the baselinesamples, and grouped the patients in two categories: those with (n=11),and those without (n=7) the presence of an ‘Interferon-signature’ atbaseline, i.e. the spontaneous over-expression of genes induced by typeI interferon (the IFN-induced genes were identified experimentally,based on microarray analyses of IFN-stimulated control PBMC). Notsurprisingly, dsDNA titers were significantly higher in the patientswith the signature (mean+/−SEM: 131.1.+/−50.1 UI/ml), compared to thepatients without (mean+/−SEM: 44.7+/−33.3, p=0.006 by Mann-Whitneytest). Measurable anti-IFNα antibodies were found in 8 follow-up samplesof the 8 patients with an IFN signature at baseline who received theimmunogenic product, while this was only the case in 2 out of 6 patientswithout IFN signature treated with the immunogenic product, and none outof the 4 placebo-treated individuals (p=0.002 by Chi-squared test). Outof the 11 patients with a baseline IFN-signature, 2 received the 30 mcgdose, 1 received the 60 mcg dose, 5 received the 120 mcg dose and 3 weretreated with a placebo injection. The changes observed in the expressionof the IFN-induced genes between V1 and V6 were significantly differentin the patients treated with the immunogenic product, as compared to thepatients treated with the placebo (FIG. 2).

This result suggests that the immunogenic product has an effect on theexpression of IFN-induced genes in vivo.

Example 9 IFN Signature and Response to Immunogenic Product-Treatment

The “Interferon-signature” at baseline was measured in the 28 patientswith mild to moderate SLE of Example 7. The Interferon-signature(IFN-signature) corresponds to a score calculated using 21 IFN-inducedgenes, and was described in Yao et al, Arthritis & Rheumatism, 2009, 60(6): 1785-1796. Measurement of the Interferon-signature was done asdescribed in Example 8.

On the 28 SLE patients of Example 7, 19 showed a positiveInterferon-signature and 9 a negative Interferon signature at baseline.

Interferon Signature and SLE Disease Activity

dsDNA antibody titers and serum levels of C3 were measured as indices ofdisease activity in both groups of patients.

dsDNA Antibody titers were determined using DPC Anti-DNA kit (PIKADD-4)from Diagnostic Products Corporation.

C3 serum levels were determined using Complement C3 kit (Kit #446450)from Beckman Coulter.

TABLE 6 Indices of SLE disease activity IFN signature IFN signaturepositive SLE negative SLE p value dsDNA Ab 147 ± 57 44 ± 93 <0.05 C3 834± 55 1199 ± 114  <0.005

As shown in the Table 6 above, SLE patients with positiveInterferon-signature at baseline have biological indices of higherdisease activity.

Interferon-Signature and Response to Treatment with the ImmunogenicProduct of the Invention

The effects of the treatment with the immunogenic product of theinvention as described in Example 7 were compared in SLE patients withpositive and negative IFN-signature at baseline.

Anti-IFN-Alpha Response

IFN-binding antibody titers were measured as described in Example 7 atV6 (day 38), V10 (day 112) and V11 (day 168 after immunization).

The results showed that SLE patients with positive Interferon-signatureproduce ten folds more IFN-binding antibodies in response to theimmunogenic product of the invention than SLE patients with negativeInterferon-signature at baseline (FIG. 3).

IFN-Induced Genes

The evolution of the expression of IFN-induced genes between V0 and V10or V11 was measured in treated patients with positive or negative IFNsignature at baseline and in patients treated with placebo.

The results showed that compared to placebo and IFN-signature negativepatients, the effects of therapy with the immunogenic product of theinvention on IFN-induced genes were strongly and significantly differentat V10 and V11 in IFN-signature positive SLE patients (FIG. 4).

Complement C3

Serum C3 values were measured in treated patients and in placeboreceiving patients at V-1 (30 days before immunization), V0 (day ofimmunization), V7 (day 56), V10 (day 112) and V11 (day 138 afterimmunization) as hereinabove described.

Results showed that there is a significant increase in C3 levels intreated—versus placebo patients. Moreover, there is a significantincrease in C3 levels in IFN-signature positive SLE patients treatedwith the immunogenic product of the invention (FIG. 5).

Example 10 Determination of the Ratio IFNα/KLH in the Product of theInvention

In order to assess the amount of IFNα and KLH in the product of theinvention, a method of quantification based on UV and fluorescencedetection was developed. The products of the invention were manufacturedwith two fluorescent labels, each specific of IFNα or KLH. Afteranalysis by Size Exclusion Chromatography (SEC), quantification of IFNαand KLH was determined by integration of the UV signal at 220 nm andfluorescent signal (FLD) specific for IFNα label or KLH label. Thismethod allowed calculating the ratio in weight of IFNα/KLH.

a) Raw Materials Labeling:

Fluorescent tags were coupled on sulfhydryl groups in order to preserveamino groups used during the product manufacturing.

Labeling was conducted in 70 mM pH7 sodium phosphate buffer at roomtemperature during 3 h. KLH were labeled with 200 molar equivalent ofAtto565-maleimide (18507, Sigma) and IFNα with 100 molar equivalent offluorescein maleimide (46130, Pierce). The labeled proteins (KLH-atto565and IFN-Fluorescein) were then filtrated on Zeba column (cut off 7 kDa,Thermo Scientific, 89893) conditioned with 70 mM pH 7.8 phosphate bufferin order to eliminate unreacted tags.

b) Product Manufacturing:

The labeled raw materials were then used to manufacture labeled productswith the same process as in Example 1 with dialysis filtration insteadof tangential flow filtration.

c) KLH and IFNα Homopolymers Standards Manufacturing

For the quantitative analytical method, homopolymers standards weremanufactured. Labeled IFNα homopolymers standard was manufactured withthe same process as in Example 1 but with 70 mM phosphate buffer pH 7.8instead of KLH and dialysis filtration instead of tangential flowfiltration.

Labeled KLH homopolymers standard was manufactured with the same processas in Example 1 but with 70 mM phosphate buffer pH 7.8 instead of IFNαand dialysis filtration instead of tangential flow filtration.

d) Method Analysis by Size Exclusion Chromatography

Batches were then analyzed by SEC with UV and specific fluorescentdetection. 60 μL of sample was injected on columns SEC5 (1000 A°) SEC3(300 A°) connected in series (Agilent, 5190-2536, 5190-2511), elutionwas performed with PBS during 35 min with UV detection at 220 nm andspecific fluorescent detection (for IFNα-Fluorescein or KLH-Atto565), asdescribed Table 7.

TABLE 7 Excitation and emission wavelength used for IFNα-Fluorescein orKLH-Atto-565 Fluorescent specific wavelength nm detection ExcitationEmission IFN_(α)-Fluorescein 490 520 KLH-Atto565 570 600

UV and fluorescent (FLD) signals were calculated by integrating the areaunder the chromatogram peaks between 0 and 20 min.

To validate this method, preliminary experiments were conducted todemonstrate:

-   -   Fluorescent signal specificity (no signal overlapping was        observed between the two labeled proteins),    -   For each manufactured batch (product of the invention, labeled        homopolymers of KLH and IFNα), similar UV profiles by SE-HPLC        were obtained,    -   No quenching of the fluorescent signal due to the manufacturing        was observed,    -   FLD signals were linear and proportional to UV signals.

Labeled IFNα UV contribution in the manufactured labeled kinoid wasmeasured according to the curve Area by FLD_(IFNα-Fluorescein)=f(Area byUV) of labeled IFNα homopolymers standard.

Labeled KLH UV contribution in the manufactured labeled kinoid wasmeasured according to the curve Area by FLD_(KLH-Atto565)=f(Area by UV)of labeled KLH homopolymers standard.

As UV area was checked to be a linear function of protein concentration,this method allowed assessing the percentage in weight of labeled IFNαin the total manufactured labeled kinoid.

e) Batches Analysis

3 batches of labeled kinoids were manufactured and analyzed by thismethod. Based on the proportionality of UV signal and concentration, andof FLD and UV signal, the ratio between the amount of IFNα and KLH(mIFNα/mKLH) was calculated for the three batches (Table 8).

TABLE 8 weight ratio of IFNα/KLH in the three labeled kinoidmanufactured Ratio m_(INF)/m_(KLH) Mean RSD % Batch 1 0.29 0.28 12 Batch2 0.31 Batch 3 0.25

A mean ratio mIFNα/mKLH of 0.28 was found with a relative standarddeviation <15%.

Example 11 Anti-mIFNα Antibodies Titers Produced and NeutralizingCapacities when Immunogenic Product of the Invention is Injected as anEmulsion with SWE or SWE-a

Manufacturing muIFN-K:

Briefly, murine IFNαA (PBL Biomedical Laboratories) and native KLH(Sigma) were mixed at a 50:1 ratio and treated with 22.5 mMglutaraldehyde for 45 minutes. After dialysis against phosphate-bufferedsaline (PBS) to eliminate excess glutaraldehyde, the solution wasincubated with 66 mM formaldehyde for 48 hours at 37° C. After quenchingwith glycine (0.1 M final) and subsequent dialysed against PBS using a10-kDa cutoff membrane, the preparation was filter-sterilized using a0.22-μm membrane and stored at 4° C.

Immunization Protocol:

Mice were immunized i.m. twice at day 0 and day 21 with mIFN-K (10 μgper injection) as an emulsion 1 to 1 with SE or SE-a adjuvant (100 μlfinal volume).

Determination of Anti-muIFNα and Anti-KLH Antibody Titers by ELISA

Sera were analyzed for antibodies against muIFNα or KLH by ELISA.Briefly, 96-well Maxisorp plates (Nunc) were coated with 100 ng/well ofmuIFNαA (PBL Biomedical Laboratories) to detect anti-muIFNα antibodiesor native KLH (Sigma) to detect anti-KLH antibodies.

Two-fold serial serum samples dilution (from 1:100 to 1:51,200) wereadded to the wells. Blank wells received 100 μL of dilution buffer.After 1.5 hours at 37° C., antibodies were detected with 100 μL ofhorseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) andO-phenylenediamine, a colorimetric substrate for horseradish peroxidase.A pool of sera from muIFN-K immunized Balb/c mice was used as a positivecontrol. The optical density (OD) was recorded at a wavelength of 490nm. ELISA assays were performed in duplicate. In each plate, two wellswere reserved for blanks; their mean value was subtracted from allwells.

Antibody titers were calculated by interpolating the maximum OD(ODmax)/2 on the x-axis. The equation used was y=ax+b for a straightline passing through two points surrounding the ODmax/2.

Determination of Neutralizing Capacity of Anti-muIFNα Antibodies Inducedafter IFN-K Immunizations

Neutralizing capacity was determined using the classical antiviralcytopathic assay (EMCV/L929). In this assay, the antiviral activitytiter of muIFNα is determined regarding its capacity to inhibit thelethal effect of encephalomyocarditis virus (EMCV) on murine L-929 cells(ATCC).

Briefly, 25 μL of diluted serum samples (or control antibody) were addedto 96-well culture plates (Nunc) in two-fold serial dilutions (from1:200 to 1:6400). A commercial rabbit polyclonal antibody anti-muIFNα(from PBL, ref: 32100-1) was used as a positive control. Afterincubation with 25 IU/well of muIFNα for 1 hour at room temperature,20×103 L-929 cells were seeded per well and incubated at 37° C. Afterovernight growth, plates were washed with PBS and 100 μL/well of EMCVsolution (100 times the dose needed to kill 50% of the cells) was addedto each well. Plates were incubated during 48 hours at 37° C. Finally,20 μL per well of MTS/PMS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner sal/phenazine methosulfate) solution (Promega) was added and theplates were incubated for 4 h at 37° C., 5% CO2 in a humidifiedincubator (protected from light). Next, the OD at 490 nm was measuredfor each well. The OD of the blank (wells with 100 μL of culture mediumalone) was subtracted from the sample OD.

The neutralizing capacity of each sample was calculated as following:

Neutralizing capacity (%)=100×[(OD test−OD virus)/(OD cells)], where

ODtest is the OD for the tested sample (cells+IFNα+serum+virus)

ODvirus is the OD for the virus control (cells+virus)

ODcells is the OD for 20,000 cells/well (cells+IFNα+virus).

Neutralizing capacities were plotted as a function of serum dilution.The titer (number of serum dilution) neutralizing 50% of IFNα activityvalues were determined by interpolation on the linear part of the curve.

Results showed that anti-muIFNα titers and anti-KLH titers were presentin mice sera collected at day 31 after first injection of muIFN-Kemulsified in SWE or SWE-a; and that the anti-muIFNα antibodies hadneutralizing capacities (NC50>200).

1-3. (canceled)
 4. A method for treating an IFNα related condition in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of an immunogenic product comprisingIFNα coupled to a carrier protein molecule, wherein said therapeuticallyeffective amount is more than 30 mcg of immunogenic product peradministration.
 5. The method according to claim 4, wherein theadministration of the therapeutically effective amount of theimmunogenic product prevents the occurrence of symptoms of a diseaselinked to an over-production of IFNα.
 6. The method according to claim4, wherein the administration of the therapeutically effective amount ofthe immunogenic product prevents the flare of a disease linked to anover-production of IFNα.
 7. The method according to claim 4, wherein theIFNα related condition is selected from the group comprising systemiclupus erythematosus, rheumatoid arthritis, scleroderma, Sjögrensyndrome, vasculitis, HIV, type I diabetes, autoimmune thyroiditis andmyositis.
 8. The method according to claim 4, wherein thetherapeutically effective amount of the immunogenic product is from 35mcg to 1000 mcg of immunogenic product per administration.
 9. The methodaccording to claim 4, wherein the immunogenic product is administratedto the subject at least twice in a month.
 10. The method according toclaim 9, wherein the immunogenic product is further administrated to thesubject at least once every three months.
 11. The method according toclaim 9, wherein the immunogenic product is further administrated to thesubject when, in a serum sample obtained from the subject, the amount ofanti-IFNα antibodies is undetectable.
 12. The method according to claim4, wherein the immunogenic product is strongly inactivated, which meansthat the product shows less than 5% of antiviral activity in theconditions of TEST B.
 13. The method according to claim 4, wherein theimmunogenic product is capable of neutralizing the antiviral activity ofIFNα in the conditions of TEST C.
 14. The method according to claim 4,wherein the immunogenic product comprises at least one subtype of IFNα.15. The method according to claim 4, wherein the immunogenic productcomprises the subtype IFNα 2b of IFNα and wherein the carrier proteinmolecule is KLH.
 16. The method according to claim 4, wherein theimmunogenic product is a vaccine, preferably in the form of an emulsion.17. A unit dosage form comprising more than 30 mcg of an immunogenicproduct comprising IFNα coupled to a carrier protein molecule.
 18. Amedical device comprising more than 30 mcg of an immunogenic productcomprising IFNα coupled to a carrier protein molecule.
 19. A kitcomprising at least one vial containing more than 30 mcg of animmunogenic product comprising IFNα coupled to a carrier proteinmolecule, at least one vial containing adjuvant, and means forcontacting said immunogenic product to the adjuvant, and for emulsifyingthe mixture with the adjuvant.